Coxiella burnetii actively express effectors that likely remodel the host cell for replication and are essential for pathogenesis are likely type 4 secretion system-dependent (T4SS), similar to Dot/Icm secretion in Legionella pneumophila. Identification of T4SS substrates of C. burnetii has been facilitated using Legionella, but studies in C. burnetii have not been done. Impediments include the obligate intracellular requirement for cultivation and lack of genetic tools. Recent advances resolving this include growth medium allowing replication outside of host cells, transposon mutagenesis and a stable shuttle vector. Objectives are to identify T4SS-dependent effectors by:1) Develop genetics to determine the requirement of T4SS. A dominant negative mutation in dotB(S162L) will be delivered to C. burnetii through either Himar1 transposon or plasmid. Clones will be compared for impact on replication in vitro and in vivo. A secretion assay in C. burnetii using either TEM1 b- lactamase or SidC fusion expression during intracellular infection will use the mutant as an important control. 2) Identify novel T4SS-dependent effector proteins. A bacterial 2-hybrid screen will identify candidates that will be verified using L. pneumophila DotA-dependent CyaA fusion secretion. Each candidate will be tested as T4SS substrates in C. burnetii. 3) Establish effector function for T4SS substrates. ORFs will be ectopically expressed in Vero cells to localize specific interactions. Antibody will be generated to localize distribution when expressed by C. burnetii. Candidates will be analyzed by pull-down and immunoprecipitation assay of cell extracts to proteomically (MS-MS) identify potential binding partners. A selected group of substrates will be tested by yeast 2-hybrid expressing a Vero cell cDNA library. A yeast toxicity model will test the ability of each putative T4SS ORF to affect viability when heterologously expressed. 4) Determine a role in virulence for T4SS substrates in vitro and in vivo using knockout mutagenesis. PUBLIC HEALTH RELEVANCE: It is our expectation that these studies will result in identification of several novel T4SS dependent effector molecules, including both acute and chronic pathotype- specific determinants. The ability to perform these studies only recently became possible with advancement in cultivation and mutagenesis procedures for C. burnetii. A comprehensive study including knockout and dominant negative expression will be unique approaches and provide a depth of study unparalleled with the agent. These data will be used to expand out understanding of the pathogenic process in Q fever and identify targets for anti-C. burnetii therapy.